利用 CRISPR-Cas9 工具在酿酒酵母中表达烟曲霉壳聚糖酶

IF 4.3 3区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Qingshuai Zhang, Hui Cao
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引用次数: 0

摘要

壳寡糖(COS)因其优异的特性而应用广泛。壳聚糖酶对壳聚糖的酶水解被认为是生产 COS 的有利途径。壳聚糖酶的异源表达前景广阔,但使用常用的大肠杆菌和Pichia pastoris菌株进行的研究在后续处理和工业可扩展性方面遇到了挑战。在这项研究中,我们选择使用安全的酵母菌株 Saccharomyces cerevisiae(GRAS),省去了甲醇诱导,从而成功地进行了表达。最终,利用 GTR-CRISPR 编辑系统,摇瓶酶活性达到 2 U/ml 。壳聚糖酶的最佳活性是在 55℃、pH 值为 5 的条件下实现的,在 30 至 50℃之间具有良好的稳定性。经过 2 小时的催化反应,产物主要由壳聚糖到壳三糖组成,主要位于壳三糖位置,在酶水解的后期阶段观察到壳聚糖含量略有增加。研究结果肯定了通过酿酒酵母表达异源壳聚糖酶的可行性,并强调了其巨大的工业潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools

Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools

Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.

Graphical Abstract

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来源期刊
Bioresources and Bioprocessing
Bioresources and Bioprocessing BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
7.20
自引率
8.70%
发文量
118
审稿时长
13 weeks
期刊介绍: Bioresources and Bioprocessing (BIOB) is a peer-reviewed open access journal published under the brand SpringerOpen. BIOB aims at providing an international academic platform for exchanging views on and promoting research to support bioresource development, processing and utilization in a sustainable manner. As an application-oriented research journal, BIOB covers not only the application and management of bioresource technology but also the design and development of bioprocesses that will lead to new and sustainable production processes. BIOB publishes original and review articles on most topics relating to bioresource and bioprocess engineering, including: -Biochemical and microbiological engineering -Biocatalysis and biotransformation -Biosynthesis and metabolic engineering -Bioprocess and biosystems engineering -Bioenergy and biorefinery -Cell culture and biomedical engineering -Food, agricultural and marine biotechnology -Bioseparation and biopurification engineering -Bioremediation and environmental biotechnology
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